Serologic and molecular studies to identify neonatal alloimmune neutropenia in a cohort of 10,000 neonates

Abstract

Summary Neonatal alloimmune neutropenia (NAIN) is caused by maternal alloimmunisation to fetal human neutrophil antigens (HNAs). This study investigated maternal HNA/HLA alloantibodies involved with NAIN and identified the frequency of NAIN in Brazilian neonates. Neonatal neutropenia (neutrophil count < 1.5 × 109/L) was investigated in samples from 10,000 unselected neonates, resulting in 88 neutropenic newborns (NBs) and their 83 mothers. Genotyping was performed by PCR‐SSP (HNA‐1/‐4) and PCR‐RFLP (HNA‐3/‐5). Serologic studies were performed by GAT (granulocyte agglutination test), Flow‐WIFT (white blood cells immunofluorescence test) and LABScreen‐Multi‐HNA‐Kit (OneLambda®) (LSM). Neonatal neutropenia was identified in 88/10,000 (0·9%) NBs. Genotyping revealed 60·2% maternal‐fetal HNA incompatibilities (31·8% for HNA‐1; 14·8% for HNA‐3; 15·9% for HNA‐4; 21·6% for HNA‐5). Serologic studies revealed 37·3% of mothers with positive results with at least one technique. The detected anti‐HNA specificities were confirmed in eight positive cases related to HNA‐1/‐3 systems. In cases with maternal‐fetal HNA‐4/‐5 incompatibility, no specific neutrophil alloantibodies were found but anti‐HLA I/II were present. Anti‐HNA‐2 was not identified. This is a large Brazilian study which involved the investigation of antibodies against all five HNA systems in neutropenia cases and showed a frequency of NAIN in 8/10,000 neonates. Among the HNA antibodies identified, we highlight the anti‐HNA‐1d and anti‐HNA‐3b, antibodies unusual in alloimmunised women, and rarely related to NAIN cases.

SLC44A2 Frequency, a New TaqMan Real-Time Polymerase Chain Reaction Method for HNA-3A/3B Genotyping, and a New Application of Droplet Digital PCR

“…Antibodies to HNA-3A can be produced following exposure to HNA-3A antigen, due to alloimmunity between the pregnant woman and the fetus or to blood transfusion ( Bux, 2008 ). The incidence of neutropenia in neonates with HNA-3 is 14.8% ( Abbas et al, 2021 ). HNA-3 is also responsible for 0.1% of the incidence of TRALI ( Roubinian, 2018 ).…”

A novel immunocomplex capture fluorescence assay (ICFA) using fluorescent beads and transfected cells for specific identification of human neutrophil antigen (HNA)‐1a and ‐1b antibodies

“…The recently developed fluorescent bead method, LABM, is widely used for HNA antibody testing. 6,10,11 However, falsepositive LABM reactions are common due to denaturation of bead-bound HNA antigens, and some antibodies cannot be detected. 4,6,12 Therefore, the ISBT and IGIW recommend the MAIGA assay, which detects antibodies in a molecule-specific manner, as the gold standard for determining HNA antibody specificity.…”

“…Our data suggest that the novel ICFA using CD16 mAb‐conjugated Luminex beads and HNA‐1‐expressing cells is highly sensitive and exhibits specificity similar to that of the MAIGA and LABM assays for detecting antibodies against HNA‐1a and HNA‐1b. The recently developed fluorescent bead method, LABM, is widely used for HNA antibody testing 6,10,11 . However, false‐positive LABM reactions are common due to denaturation of bead‐bound HNA antigens, and some antibodies cannot be detected 4,6,12 .…”

Combined Neonatal Alloimmune Neutropenia and Thrombocytopenia in Dizygotic Twins Conceived After Oocyte Donation

“…Incidence of NAIT has been estimated at 1 in 1000 to 10000 live births [5][6][7], while the incidence of NAIN has been reported to be between 2/1000 to 8/10000 live births (some studies have estimated lower rates up to 1/120000) [2,[8][9][10][11].…”